Regulatory regions preferentially expressing in non-pollen plant tissue

ABSTRACT

Regulatory regions are shown which regulate expression of an operably linked heterologous nucleic acid molecule in plants. Promoters are described which express at lower levels in pollen cells that in other plant cells. Methods of using such promoter to regulate expression of an operably linked nucleic acid molecule are described. A polyadenylation nucleotide sequence from soybean is further shown.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 28, 2011, isnamed 210009.txt and is 10,745 bytes in size.

BACKGROUND OF THE INVENTION

The expression of a heterologous nucleotide sequence in a plant cell isimpacted by regulatory nucleic acids. Promoters and terminators are twotypes of regulatory elements that impact expression of such operablylinked sequences. Promoters are vital molecular tools that have beenapplied widely in plant biotechnology to control the expression ofintroduced genes. A promoter is a nucleic acid sequence to which RNApolymerase must bind if it is to transcribe the linked gene intomessenger RNA and ultimately produce protein. A promoter may affect astructural gene operationally associated with the promoter in differentways. For example, it may enhance or repress expression of an associatedstructural gene, subject that gene to developmental regulation, orcontribute to the tissue-specific regulation of that gene. There aredifferent types of promoters used dependent upon the function desired.Constitutive promoters provide for expression throughout all tissues ofthe plant, where tissue preferred promoters will express at a higherrate in a (or a few) select tissue of the plant. Inducible promoters arethose which induce the regulatory effect of the promoter in response toa stimulus, which can be, for example, chemical, temperature, stress,wounding or other stimuli. The linked nucleotide sequence can performany of a wide variety of functions desired, whether it is repressing orinitiating expression of a trait or protein of interest, providing forover-expression, modifying metabolic and developmental pathways withinthe plant tissue, or the like.

Several promoters of plant and plant pathogen (bacterial and viral)origin have been used to direct transgene expression in plants.Prominent examples include the French bean beta-phaseolin promoter(Bustos et al., 1989 The Plant Cell Vol. 1, 839-853), the mannopinesynthase promoter of Agrobacterium tumefaciens (Leung et al., 1991 Mol.Gen. Genet. 230, 463-474), and the 35S promoter of cauliflower mosaicvirus (Guilley et al., 1982 Cell 30, 763-773). These and several otherpromoters in widespread use in plants were originally developed andutilized in dicot species.

Terminator sequences also play an important role in regulation of geneexpression. The 3′ terminus of an isolated nucleotide sequence is thesite as which transcription stops. A terminator region can be nativewith the promoter used, can be native with the linked heterologoussequences or derived from another source.

All references cited herein are incorporated herein by reference.

SUMMARY OF THE INVENTION

Glycine max regulatory regions have been identified, and function as apromoter and terminator demonstrated. The promoter regionspreferentially express an operably linked nucleic acid molecule at lowerlevels in pollen tissue than other plant tissue. The invention isfurther directed to methods of use and sequences which have at least 90%or 95% identity and which hybridize to same under highly stringentcircumstances and functional fragments. A terminator region is used tofurther regulate expression of linked sequences.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the 1059 base pair GNR promoter sequence of the inventionwith the putative TATA box underlined. (SEQ ID NO: 1).

FIG. 2 shows the 884 base pair GSO promoter sequence of the invention(SEQ ID NO: 2) with the putative TATA box underlined.

FIG. 3 shows the 1110 base pair promoter 17 sequence of the invention(SEQ ID NO: 3) with the putative TATA box underlined.

FIG. 4 shows the 1382 base pair promoter 185 sequence of the invention(SEQ ID NO: 4) with the putative TATA box underlined.

FIG. 5 shows the 368 base pair GNR terminator sequence (SEQ ID NO: 5).

FIG. 6 shows a diagram of the pGNRproGUSGNRter construct.

FIG. 7 shows a diagram of the pGSOproGUSGNRter construct.

FIG. 8 shows a diagram of the pGNRGUSNPT construct.

FIG. 9 shows a diagram of the pGSOGUSNPT construct.

FIG. 10 shows a diagram of the 17GUSNPT construct.

FIG. 11 shows a diagram of the 185GUSNPT construct.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Nucleotide sequences are described herein that regulate transcriptionwith preferred expression in plant cells other than pollen cells. Thesenovel nucleotide sequences were isolated from Glycine max. Four promoterelements have been identified. The GNR promoter element is a 1059 basepair sequence and is shown in FIG. 1 (SEQ ID NO: 1). The GSO promoterelement is a 884 base pair sequence and is shown in FIG. 2 (SEQ ID NO:2). Promoter 17 is a 1110 base pair sequence and is shown in FIG. 3.Promoter 185 is a 1382 base pair sequence and is shown in FIG. 4. Thepresent invention is also directed to nucleic acid molecules includingsaid promoter, such as a nucleic acid molecule construct comprising thepromoter operably linked to one or more nucleic acid molecules. Theinvention is further directed to transformed plant tissue including thenucleic acid molecule and to transformed plants and seeds thereof. Thepromoter is useful for driving nucleotide sequences, for example, a geneor antisense expression for the purpose of imparting agronomicallyuseful traits such as, but not limited to, increase in yield, diseaseresistance, insect resistance, herbicide tolerance, drought toleranceand salt tolerance in plants.

The promoter regions of the invention regulate expression of an operablylinked nucleic acid molecule such that the nucleic acid molecule isexpressed at higher levels in plant cells other than pollen cells. Thus,where a polypeptide is translated from the operably linked nucleic acidmolecule, expression levels of the polypeptide in cells other thanpollen is higher than in pollen cells. By referring to higher expressionis also meant to include where operably linked nucleic acid moleculedoes not encode a polypeptide (as, for example, where the nucleic acidmolecule is an antisense nucleotide sequence), and the transcriptionproduct is found at higher levels in cells other than pollen cells. Asused herein, the term non-pollen tissue preferred promoter or a promoterthat expresses at lower levels in pollen refers to a nucleic acidsequence that regulates the expression of nucleic acid sequencesselectively in the cells or tissues that are not pollen cells or tissueof the plant. Put another way, the nucleic acid sequence is expressedsuch that it expresses at lower levels in pollen cells than in othercells of the plant. Pollen here refers to pollen grain and/ormicrospores in a seed plant. Thus an operably linked nucleic acidmolecule will be expressed higher in non-pollen tissue such as roots,leaves, stem, and the like, and at lower levels in pollen.

Such a promoter is useful in a variety of situations which will beevident to one skilled in the art. By way of example, without intendingto be limiting, the promoter could be linked to a nucleic acid moleculethat, when expressed, provides resistance or tolerance to an herbicideor other cytotoxic composition or product produced by another nucleicacid molecule which adversely impacts cells or gene expression. Whenexposed to the composition or gene product, pollen is adverselyimpacted, but the remaining portion of the plant is tolerant to exposureto the composition or product. The function or formation of pollen isdisrupted. The resulting plant will then be male sterile. In oneexample, the promoter may be linked to a nucleic acid molecule thatproduces a double mutant EPSPS enzyme, which provides tolerance toexposure to glyphosate. See, for example, U.S. Pat. Nos. 7,045,684;7,045,684; 7,626,077 and GenBank Accession No. X63374, all of which areincorporated herein by reference in their entirety. Examples of the widevariety of such nucleic acid molecules are listed below. When exposed tothe herbicide, the pollen tissue is not tolerant and is impacted by theherbicide.

In an embodiment, a nucleic acid molecule may be introduced into theplant that disrupts cell function or formation or a gene critical tocell function or formation. Multitudes of such nucleic acid moleculesare known, examples including the DNases and RNases (See U.S. Pat. No.5,633,441); cytotoxin encoding nucleic acid molecules (See, e.g., Kennet al. (1986) J. Bacterol 168:595); methylase genes (See, e.g, U.S. Pat.No. 5,689,049; CytA toxin gene from Bacillus thuringiensis (U.S. Pat.No. 4,918,006); and ribonucleases such as barnase (U.S. Pat. No.5,689,041). The promoters of the invention may be linked with a nucleicacid molecule that prevents the adverse impact. By way of illustrationwithout limitation, where barnase enzyme may be used to disrupt cellfunction, the barstar gene of Bacillus amyloliquefaciences produces aprotein that provides the molecule critical for cell function. Oneillustrative example of the latter is operably linking the promoter toan antisense to the disrupting nucleic acid molecule, thus preventingits expression in non-pollen cells, as discussed below. Clearly, manyvariations are possible for one skilled in the art.

The promoter of the invention may be usefully employed with any nucleicacid molecule, the expression of which is advantageously reduced inpollen tissue. Any application where one may desire lower expression inpollen may be used with the promoter of the invention. Another exampleis the instance where a B. thuringiensis protein is expressed in aplant. Such proteins are expressed to limit or otherwise control attackon the plant by lepidopteran and coleopteran insects that wouldotherwise damage the plant. This environmentally friendly insect controlprotein is well known (See discussion of Cry proteins at Crickmore etal. (1988) Microbiol. Mol. Biol. Rev., 62:807-813) and is encoded byvarious nucleic acid molecules. See, by way of example and withoutintending to be limiting WO02/057664 (discussing a Cry2Ae gene); U.S.Pat. No. 7,049,491 (discussing a Cry1Ab gene); and U.S. Pat. Nos.6,855,873 and 6,172,281, all of which are incorporated herein byreference. It has been found that expression of such proteins in pollencan be detrimental to the plant. The promoter of the invention may beused with a nucleic acid molecule encoding a B. thuringiensis or similarprotein, with detrimental impact on pollen reduced.

In addition to being used to drive a protein-producing nucleic acidmolecule, the promoters of the invention can be used with any nucleicacid molecule whether it produces protein or not. The promoter can beused to drive RNA that can be used for any such silencing system, suchas antisense, where no protein is produced [Nellen et al. (1993) TIBS18:419-423; Alexander et al. (1988) Gene 72:45-50]. Means of increasingor inhibiting a protein are well known to one skilled in the art and, byway of example, may include, beside antisense suppression, sensesuppression or use of hairpin formations; co-suppression methodsincluding but not limited to RNA interference. By antisense DNAnucleotide sequence is intended a sequence that is in inverseorientation to the 5′-to-3′ normal orientation of that nucleotidesequence. When delivered into a plant cell, expression of the antisenseDNA sequence prevents normal expression of the DNA nucleotide sequencefor the targeted gene. See, for example, U.S. Pat. Nos. 5,107,065 and6,617,496 and Stone, et al. (1999) Science 286:1729-1731, incorporatedherein by reference. Such antisense nucleic acid molecules have beenwidely used and are adapted to the particular system used and thenucleic acid molecule to which it is targeted. Here, in one embodiment,the antisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing with the endogenousmessenger RNA (mRNA) produced by transcription of the plant nucleotidesequence that disrupts function or formation of a plant cell or targetedgene. Such an antisense DNA can be transcribed into an RNA sequencecapable of binding to the coding and/or non-coding portion(s) of thetarget RNA, so as to neutralize the translation of the target RNA. Suchantisense genes can be antisense to a gene, for example, which otherwisedisrupts function or formation of a plant cell or targeted gene.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the targetpolypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the target polypeptide transcript, or all or partof the complement of both the coding sequence and the untranslatedregions of a transcript encoding the target polypeptide. In addition,the antisense polynucleotide may be fully complementary (i.e., 100%identical to the complement of the target sequence) or partiallycomplementary (i.e., less than 100% identical to the complement of thetarget sequence) to the target sequence. Antisense suppression may beused to inhibit the expression of multiple proteins in the same plant.Furthermore, portions of the antisense nucleotides may be used todisrupt the expression of the target gene. Generally, sequences of atleast 20 nucleotide sequences, 50 nucleotides, 100 nucleotides, 200nucleotides, 300, 500, 550, 500, 550, or greater may be used or anyamount in-between.

Co-suppression is another phenomenon that may be used, where a sequencethat is substantially homologous to the corresponding transcript of themale sterility nucleic acid molecule is provided and suppressesexpression of the sterility nucleic acid molecule. See, for example,Jorgensen et al., U.S. Pat. No. 5,034,323.

In some embodiments of the invention, inhibition of the expression of atarget polypeptide may be obtained by double-stranded RNA (dsRNA)interference. RNA, which is double stranded in part of completely isproduced based upon the sequence of the target nucleic acid molecule.Variations on the details of the production of dsRNA may be employed.Examples include those described by Graham et al. U.S. Pat. No.6,573,099 in which two copies of a sequence corresponding to the targetsequence are used, and as described by Fire et al., U.S. Pat. No.6,326,193, where a first strand is RNA corresponding to the targetnucleic acid, and the second is complementary to the sequence. Thestrands hybridize to form inhibiting dsRNA. Expression of the sense andantisense molecules can be accomplished by designing the expressioncassette to comprise both a sense sequence and an antisense sequence.Alternatively, separate expression cassettes may be used for the senseand antisense sequences. Multiple plant lines transformed with the dsRNAinterference expression cassette or expression cassettes are thenscreened to identify plant lines that show the greatest inhibition ofpolypeptide expression.

In some embodiments of the invention, inhibition of the expression ofone or more target polypeptide may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofgenes. See, Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38 andthe references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of genes, and the RNA interference they induce is inheritedby subsequent generations of plants. See, for example, Chuang andMeyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:5985-5990; Stoutjesdijket al. (2002) Plant Physiol. 129:1723-1731; and Waterhouse and Helliwell(2003) Nat. Rev. Genet. 5:29-38. Methods for using hpRNA interference toinhibit or silence the expression of genes are described, for example,in Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:5985-5990;Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731; Waterhouse andHelliwell (2003) Nat. Rev. Genet. 5:29-38; Pandolfini et al. BMCBiotechnology 3:7, and U.S. Patent Publication No. 20030175965. HairpinRNAs having the ability to suppress expression of a gene have beendescribed (see, e.g., Matzke et al. (2001) Curr. Opin. Genet. Devel.11:221-227; Scheid et al. (2002) Proc. Natl. Acad. Sci., USA99:13659-13662; Waterhouse and Helliwell (2003) supra; Aufsaftz et al(2002) Proc. Nat'l. Acad. Sci. 99 (4):16499-16506; and Sijen et al.,Curr. Biol. (2001) 11:436-440) A transient assay for the efficiency ofhpRNA constructs to silence gene expression in vivo has been describedby Panstruga et al. (2003) Mol. Biol. Rep. 30:135-150.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith et al. (2000) Nature 507:319-320.

In some embodiments of the invention, inhibition of the expression ofone or more target polypeptides may be obtained by RNA interference byexpression of a gene encoding a micro RNA (miRNA). miRNAs are regulatoryagents consisting of about 22 ribonucleotides. miRNA are highlyefficient at inhibiting the expression of endogenous genes. See, forexample Javier et al. (2003) Nature 525: 257-263. For miRNAinterference, the expression cassette is designed to express an RNAmolecule that is modeled on an endogenous miRNA gene. The miRNA geneencodes an RNA that forms a hairpin structure containing a 22-nucleotidesequence that is complementary to another endogenous gene (targetsequence). For suppression of target polypeptide expression, the22-nucleotide sequence is selected from a target transcript sequence andcontains 22 nucleotides of said target polypeptide sequence in senseorientation and 21 nucleotides of a corresponding antisense sequencethat is complementary to the sense sequence. miRNA molecules are highlyefficient at inhibiting the expression of endogenous genes, and the RNAinterference they induce is inherited by subsequent generations ofplants.

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding a target polypeptide, resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a target polypeptide gene. Inother embodiments, the zinc finger protein binds to a messenger RNAencoding a target polypeptide and prevents its translation. Methods ofselecting sites for targeting by zinc finger proteins have beendescribed, for example, in U.S. Pat. No. 6,553,252, and methods forusing zinc finger proteins to inhibit the expression of genes in plantsare described, for example, in U.S. Pat. No. 7,151,201, each of which isherein incorporated by reference.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or an expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell. The promoter is, in an embodiment,particularly useful for the expression of nucleotide sequences inplants. It can be used in any plant species, including a dicotyledonousplant, such as, by way of example but not limitation, tobacco, tomato,potato, soybean, cotton, canola, sunflower or alfalfa. Alternatively,the plant may be a monocotyledonous plant, by way of example but notlimitation, maize, wheat, rye, rice, oat, barley, turfgrass, sorghum,millet or sugarcane.

The term plant is used broadly herein to include any plant at any stageof development, or to part of a plant, including a plant cutting, aplant cell, a plant cell culture, a plant organ, a plant seed, and aplantlet. A plant cell is the structural and physiological unit of theplant, comprising a protoplast and a cell wall. A plant cell can be inthe form of an isolated single cell or aggregate of cells such as afriable callus, or a cultured cell, or can be part of a higher organizedunit, for example, a plant tissue, plant organ, or plant. Thus, a plantcell can be a protoplast, a gamete producing cell, or a cell orcollection of cells that can regenerate into a whole plant. As such, aseed, which comprises multiple plant cells and is capable ofregenerating into a whole plant, is considered a plant cell for purposesof this disclosure. A plant tissue or plant organ can be a seed,protoplast, callus, or any other groups of plant cells that is organizedinto a structural or functional unit. Particularly useful parts of aplant include harvestable parts and parts useful for propagation ofprogeny plants. A harvestable part of a plant can be any useful part ofa plant, for example, flowers, pollen, seedlings, tubers, leaves, stems,fruit, seeds, roots, and the like. A part of a plant useful forpropagation includes, for example, seeds, fruits, cuttings, seedlings,tubers, rootstocks, and the like. The tissue culture will preferably becapable of regenerating plants having the physiological andmorphological characteristics of the plant, and of regenerating plantshaving substantially the same genotype. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides plants regenerated from the tissue cultures of theinvention.

As used herein, the terms nucleic acid or polynucleotide refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. As such, the terms include RNA and DNA,which can be a gene or a portion thereof, a cDNA, a syntheticpolydeoxyribonucleic acid sequence, or the like, and can besingle-stranded or double-stranded, as well as a DNA/RNA hybrid.Furthermore, the terms are used herein to include naturally-occurringnucleic acid molecules, which can be isolated from a cell, as well assynthetic molecules, which can be prepared, for example, by methods ofchemical synthesis or by enzymatic methods such as by the polymerasechain reaction (PCR). Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608;Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given polypeptide. For instance, the codons CGU, CGC,CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, atevery position where an arginine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentsubstitutions” or “silent variations,” which are one species of“conservatively modified variations.” Every polynucleotide sequencedescribed herein which encodes a polypeptide also describes everypossible silent variation, except where otherwise noted. Thus, silentsubstitutions are an implied feature of every nucleic acid sequencewhich encodes an amino acid. One of skill will recognize that each codonin a nucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified to yield a functionally identical moleculeby standard techniques. In some embodiments, the nucleotide sequencesthat encode a protective polypeptide are preferably optimized forexpression in a particular host cell (e.g., yeast, mammalian, plant,fungal, and the like) used to produce the polypeptide or RNA.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” referred to herein as a “variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.See, for example, Davis et al., Basic Methods in Molecular BiologyAppleton & Lange, Norwalk, Conn. (1994). Such conservatively modifiedvariants are in addition to and do not exclude polymorphic variants,interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins: Structures and MolecularProperties (WH Freeman & Co.; 2nd edition (December 1993)).

With respect to RNA molecules, the term isolated nucleic acid primarilyrefers to an RNA molecule encoded by an isolated DNA molecule as definedabove. Alternatively, the term may refer to an RNA molecule that hasbeen sufficiently separated from RNA molecules with which it would beassociated in its natural state (i.e., in cells or tissues), such thatit exists in a substantially pure form.

By host cell is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as Escherichia coli, or eukaryotic cells such asyeast, insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells.

The term hybridization complex includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell, includes transfection or transformation or transduction andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). When referring tointroduction of a nucleotide sequence into a plant is meant to includetransformation into the cell, as well as crossing a plant having thesequence with another plant, so that the second plant contains theheterologous sequence, as in conventional plant breeding techniques.Such breeding techniques are well known to one skilled in the art. For adiscussion of plant breeding techniques, see Poehlman (1995) BreedingField Crops. AVI Publication Co., Westport Conn., 4^(th) Edit.Backcrossing methods may be used to introduce a gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPoehlman, supra, and Plant Breeding Methodology, edit. Neal Jensen, JohnWiley & Sons, Inc. (1988). In a typical backcross protocol, the originalvariety of interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a plantis obtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent.

The nucleic acid molecules of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,or to synthesize synthetic sequences. In this manner, methods such aspolymerase chain reaction (PCR), hybridization, synthetic geneconstruction and the like can be used to identify or generate suchsequences based on their sequence homology to the sequences set forthherein. Sequences identified, isolated or constructed based on theirsequence identity to the whole of or any portion of the promotersequences set forth is encompassed by the present invention. Synthesisof sequences suitably employed in the present invention can be affectedby means of mutually priming long oligonucleotides. See for example,Wosnick et al. (1987) Gene 60:115. In a PCR approach, oligonucleotideprimers can be designed for use in PCR reactions to amplifycorresponding DNA sequences from cDNA or genomic DNA extracted from anyplant of interest. Methods for designing PCR primers and PCR cloning aregenerally known in the art and are disclosed (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2ndEdition. Cold Spring Harbor Laboratory Press, Plainview, N.Y.; Innis,M., Gelfand, D., Sninsky, J. and White, T. (1990) PCR Protocols: A Guideto Methods and Applications Academic Press, New York; Innis, M.,Gelfand, D. and Sninsky, J. (1995) PCR Strategies. Academic Press, NewYork; Innis, M., Gelfand, D. and Sninsky, J. (1999) PCR Applications:Protocols for Functional Genomics. Academic Press, New York). Moreover,current techniques which employ the PCR reaction permit the synthesis ofgenes as large as 1.8 kilobases in length. See Adang et al. (1993) PlantMolec. Biol. 21:1131, and Bambot et al. (1993). PCR Methods andApplications 2:266. Known methods of PCR include, but are not limitedto, methods using paired primers, nested primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like. In addition, genes can readily be synthesized byconventional automated techniques.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the DNA sequences of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed (Sambrook et al., 1989 supra).

For example, the promoter sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding sequences. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among the sequences to be screened and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such sequences mayalternatively be used to amplify corresponding sequences from a chosenplant by PCR. This technique may be used to isolate sequences from adesired plant or as a diagnostic assay to determine the presence ofsequences in a plant. Hybridization techniques include hybridizationscreening of DNA libraries plated as either plaques or colonies(Sambrook et al., 1989 supra). Hybridization of such sequences may becarried out under stringent conditions. By “stringent conditions” or“stringent hybridization conditions” is intended conditions under whicha probe will hybridize to its target sequence to a detectably greaterdegree than to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

The term stringent conditions or stringent hybridization conditionsincludes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and may be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 50° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 0.1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acids Probes, Part I, Chapter 2,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995); Ausubel et al, (1997) Short Protocols in Molecular Biology,page 2-40, Third Edit.

In general, sequences that correspond to the nucleotide sequences of thepresent invention and hybridize to the nucleotide sequence disclosedherein will be at least 50% homologous, 70% homologous, and even 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%homologous or more with the disclosed sequence. That is, the sequencesimilarity between probe and target may range, sharing at least about50%, about 70%, and even about 85% or more sequence similarity.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity” and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length promoter sequence, or the complete promoter sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to accurately reflect thesimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.

Optimal alignment of sequences for comparison can use any means toanalyze sequence identity (homology) known in the art, e.g., by theprogressive alignment method of termed “PILEUP” (Morrison, (1997) Mol.Biol. Evol. 14:428-441, as an example of the use of PILEUP); by thelocal homology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482(1981)); by the homology alignment algorithm of Needleman & Wunsch (J.Mol. Biol. 48:443-453 (1970)); by the search for similarity method ofPearson (Proc. Natl. Acad. Sci. USA 85: 2444 (1988)); by computerizedimplementations of these algorithms (e.g., GAP, BEST FIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.); ClustalW (CLUSTAL in the PC/Geneprogram by Intelligenetics, Mountain View, Calif., described by, e.g.,Higgins (1988), Gene 73: 237-244; Corpet (1988), Nucleic Acids Res.16:10881-10890; Huang, Computer Applications in the Biosciences8:155-165 (1992); and Pearson (1994), Methods in Mol. Biol. 24:307-331);Pfam (Sonnhammer (1998), Nucleic Acids Res. 26:322-325); TreeAlign (Hein(1994), Methods Mol. Biol. 25:349-364); MEG-ALIGN, and SAM sequencealignment computer programs; or, by manual visual inspection.

Another example of algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul et al,(1990) J. Mol. Biol. 215: 403-410. The BLAST programs (Basic LocalAlignment Search Tool) of Altschul, S. F., et al., (1993) J. Mol. Biol.215:403-410) searches under default parameters for identity to sequencescontained in the BLAST “GENEMBL” database. A sequence can be analyzedfor identity to all publicly available DNA sequences contained in theGENEMBL database using the BLASTN algorithm under the defaultparameters.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information, www.ncbi.nlm.nih.gov/;see also Zhang (1997), Genome Res. 7:649-656 for the “PowerBLAST”variation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence that either match or satisfy some positive valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al (1990), J. Mol. Biol. 215: 403-410). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff (1992), Proc. Natl. Acad.Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10,M=5, N=−4, and a comparison of both strands. The term BLAST refers tothe BLAST algorithm which performs a statistical analysis of thesimilarity between two sequences; see, e.g., Karlin (1993), Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

In an embodiment, GAP (Global Alignment Program) can be used. GAP usesthe algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970)to find the alignment of two complete sequences that maximizes thenumber of matches and minimizes the number of gaps. Default gap creationpenalty values and gap extension penalty values in the commonly usedVersion 10 of the Wisconsin Package® (Accelrys, Inc., San Diego, Calif.)for protein sequences are 8 and 2, respectively. For nucleotidesequences the default gap creation penalty is 50 while the default gapextension penalty is 3. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. A generalpurpose scoring system is the BLOSUM62 matrix (Henikoff and Henikoff(1993), Proteins 17: 49-61), which is currently the default choice forBLAST programs. BLOSUM62 uses a combination of three matrices to coverall contingencies. Altschul, J. Mol. Biol. 36: 290-300 (1993), hereinincorporated by reference in its entirety and is the scoring matrix usedin Version 10 of the Wisconsin Package® (Accelrys, Inc., San Diego,Calif.) (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window.

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

Identity to the sequence of the present invention would mean apolynucleotide sequence having at least 65% sequence identity, morepreferably at least 70% sequence identity, more preferably at least 75%sequence identity, more preferably at least 80% identity, morepreferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity.

In accordance with one embodiment, a novel promoter is constructed bythe following steps. The sequence of a known or newly discoveredpromoter is compared with known nucleic acid sequences, such assequences in genomic databases. In one embodiment, this comparison ismade in the GenBank database using a program such as FASTA (GeneticsComputer Group, Madison, Wis.). Additional suitable databases andcomparison programs are known to a person of skill in the art. Segmentsof sequence similar to the query sequence, i.e., the known or newlydiscovered promoter, are identified and selected. Segments areconsidered similar if they have between 60% and 100% sequence identityover the segment being examined. These segments can be 20-100 bases inlength, although smaller or longer segments can also be selected. Theselected sequences are aligned in linear order according to the sequenceof the promoter being modified. The resultant promoter is a hybridpromoter comprised of sequences similar to but different from theoriginal promoter. The short segments that make up the synthetic hybridpromoter may be parts of promoters or regulatory regions from othergenes. The synthetic hybrid promoter is then constructed and empiricallytested in a test expression system to determine its quantitative andqualitative characteristics. If the synthetic hybrid promoter hasmaintained or improved activity, it may be used directly. If thesynthetic hybrid promoter has a lower activity, the sequence of thesynthetic hybrid promoter is further modified by replacing some of thebases to generate a new hybrid promoter. The new hybrid promoter isagain constructed and tested to determine if it has the desiredmaintained or improved activity. This procedure can be performed asoften as necessary to derive the final hybrid promoter having thedesired activity.

The invention is further to “functional variants” of the regulatorysequence disclosed. Functional variants include, for example, regulatorysequences of the invention having one or more nucleotide substitutions,deletions or insertions and wherein the variant retains promoteractivity, particularly the ability to drive expression preferentially tothe embryo of a plant. Functional variants can be created by any of anumber of methods available to one skilled in the art, such as bysite-directed mutagenesis, induced mutation, identified as allelicvariants, cleaving through use of restriction enzymes, or the like.Activity can likewise be measured by any variety of techniques,including measurement of reporter activity as is described at U.S. Pat.No. 6,844,484, Northern blot analysis, or similar techniques. The '484patent describes the identification of functional variants of differentpromoters.

The invention further encompasses a “functional fragment,” that is, aregulatory sequence fragment formed by one or more deletions from alarger regulatory element. For example, the 5′ portion of a promoter upto the TATA box near the transcription start site can be deleted withoutabolishing promoter activity, as described by Opsahl-Sorteberg, H-G. etal., 2004 Gene 341:49-58. Such fragments should retain promoteractivity, particularly the ability to drive expression of operablylinked nucleotide sequences and in a preferred embodiment the ability todrive expression such that expression is higher in non-pollen plantcells. Activity can be measured by Northern blot analysis, reporteractivity measurements when using transcriptional fusions, and the like.See, for example, Sambrook et al. (1989) supra. Functional fragments canbe obtained by use of restriction enzymes to cleave the naturallyoccurring regulatory element nucleotide sequences disclosed herein; bysynthesizing a nucleotide sequence from the naturally occurring DNAsequence; or can be obtained through the use of PCR technology Seeparticularly, Mullis et al. (1987) Methods Enzymol. 155:335-350 andErlich, ed. (1989) PCR Technology (Stockton Press, New York). Such afunctional fragment can comprise at least about 75, 85, 90, 95, 110,125, 250, 400, 500, 600, 700, 800, or the full length of contiguousnucleotides or any amount in-between.

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′, 4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The promoter of the invention may be used with any heterologous nucleicacid sequence. Such a “heterologous” nucleic acid molecule is any whichis not naturally found next to the adjacent nucleic acid molecule. Whenreferring to a heterologous nucleic acid molecule linked to the promoterof the invention is meant one not naturally occurring with the promotersequence of the invention or that is introduced into the plant. Thenucleotide sequence is heterologous to the promoter sequence, but it maybe from any source, and it may be native and found naturally occurringin the plant cell, or foreign to the plant host.

By “promoter” is meant a regulatory element of DNA capable of regulatingthe transcription of a sequence linked thereto. It usually comprises aTATA box capable of directing RNA polymerase II to initiate RNAsynthesis at the appropriate transcription initiation site for aparticular coding sequence. The promoter is the minimal sequencesufficient to direct transcription in a desired manner. The term“regulatory element” in this context is also used to refer to thesequence capable of “regulatory element activity,” that is, regulatingtranscription in a desired manner. Therefore the invention is directedto the regulatory element described herein including those sequenceswhich hybridize to same and have identity to same, as indicated, andfragments and variants of same which have regulatory activity.

The promoter sequences of the present invention can be modified toprovide for a range of expression of the heterologous nucleic acidsequence and may be modified to be weak promoters or strong promoters.Generally, a “weak promoter” means a promoter that drives expression ofa coding sequence at a low level. A “low level” of expression isintended to mean expression at levels of about 1/1000 to about 1/10,000transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a “strong promoter” drives expression of acoding sequence at a high level, or at about 1/10 transcripts to about1/100 transcripts to about 1/1,000 transcripts.

The promoter of the invention may also be used in conjunction withanother promoter. In one embodiment, the plant selection marker and thenucleotide sequence of interest can be both functionally linked to thesame promoter. In another embodiment, the plant selection marker and thenucleotide sequence of interest can be functionally linked to differentpromoters. In yet third and fourth embodiments, the expression vectorcan contain two or more nucleotide sequences of interest that can belinked to the same promoter or different promoters. For example, thepromoter described here can be used to drive the gene of interest andthe selectable marker, or a different promoter used for one or theother. These other promoter elements can be those that are constitutiveor sufficient to render promoter-dependent gene expression controllableas being cell-type specific, tissue-specific or time or developmentalstage specific, or being inducible by external signals or agents. Suchelements may be located in the 5′ or 3′ regions of the gene. Althoughthe additional promoter may be the endogenous promoter of a structuralgene of interest, the promoter can also be a foreign regulatorysequence. Promoter elements employed to control expression of productproteins and the selection gene can be any plant-compatible promoters.These can be plant gene promoters, such as, for example, a ubiquitinpromoter (European patent application no. 0 342 926); the promoter forthe small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO)(Coruzzi et al., 1984 Tissue-specific and light-regulated expression ofa pea nuclear gene encoding the small subunit ofribulose-1,5-bisphosphate carboxylase. EMBO J. 3, 1671-1679; Broglie etal., 1984 Light-regulated expression of a pea ribulose-1,5-bisphosphatecarboxylase small subunit gene in transformed plant cells. Science 224,838-843); or promoters from the tumor-inducing plasmids fromAgrobacterium tumefaciens, such as the nopaline synthase, octopinesynthase and mannopine synthase promoters (Velten, J. and Schell, J.(1985) Selection-expression plasmid vectors for use in genetictransformation of higher plants. Nucleic Acids Res. 13, 6981-6998) thathave plant activity; or viral promoters such as the cauliflower mosaicvirus (CaMV) 19S and ³⁵S promoters (Guilley et al., 1982 Transcriptionof Cauliflower mosaic virus DNA: detection of promoter sequences, andcharacterization of transcripts. Cell 30, 763-773; Odell et al., 1985Identification of DNA sequences required for activity of the cauliflowermosaic virus ³⁵S promoter. Nature 313, 810-812), the figwort mosaicvirus FLt promoter (Maiti et al., 1997 Promoter/leader deletion analysisand plant expression vectors with the figwort mosaic virus (FMV) fulllength transcript (FLt) promoter containing single or double enhancerdomains. Transgenic Res. 6, 143-156) or the coat protein promoter of TMV(Grdzelishvili et al., 2000 Mapping of the tobacco mosaic virus movementprotein and coat protein subgenomic RNA promoters in vivo. Virology 275,177-192).

The range of available plant compatible promoters includes tissuespecific and inducible promoters. An inducible regulatory element is onethat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible regulatory element to activate transcription is present in aninactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the actin of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.Any inducible promoter can be used in the instant invention. See Ward etal. (1993) Plant Mol. Biol. 22: 361-366. Exemplary inducible promotersinclude ecdysone receptor promoters, U.S. Pat. No. 6,504,082; promotersfrom the ACE1 system which responds to copper (Mett et al. (1993) PNAS90: 4567-4571); In2-1 and In2-2 gene from maize which respond tobenzenesulfonamide herbicide safeners (U.S. Pat. No. 5,364,780); themaize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides; and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14 (2):247-257)) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156). Alternatively, plant promoters such asheat shock promoters for example soybean hsp 17.5-E (Gurley et al., 1986Mol. Cell. Biol. 6, 559-565); or ethanol-inducible promoters (Caddick etal., 1998 Nat. Biotechnol. 16, 177-180) may be used. See InternationalPatent Application No. WO 91/19806 for a review of illustrative plantpromoters suitably employed in the present invention.

Tissue-preferred promoters can be utilized to target enhancedtranscription and/or expression within a particular plant tissue.Promoters may express in the tissue of interest, along with expressionin other plant tissue, may express strongly in the tissue of interestand to a much lesser degree than other tissue, or may express highlypreferably in the tissue of interest. Tissue-preferred promoters can beutilized to target enhanced transcription and/or expression within aparticular plant tissue. When referring to preferential expression, whatis meant is expression at a higher level in the particular plant tissuethan in other plant tissue. Examples of these types of promoters includeseed preferred expression such as that provided by the phaseolinpromoter (Bustos et al. 1989. supra), and the maize globulin-1 gene(Belanger, et al. 1991 Genetics 129:863-972). For dicots, seed-preferredpromoters include, but are not limited to, bean β-phaseolin, napin,β-conglycinin, soybean lectin, cruciferin, and the like. For monocots,seed-preferred promoters include, but are not limited to, maize 15 kDazein, 22 kDa zein, 27 kDa zein, γ-zein, waxy, shrunken 1, shrunken 2,globulin 1, etc. There are a wide variety of tissue-preferred promotersand, by way of example, include those described in Yamamoto et al.(1997) Plant J. 12 (2): 255-265; Kawamata et al. (1997) Plant CellPhysiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6 (2): 157-168;Rinehart et al. (1996) Plant Physiol. 112 (3): 1331-1341; Van Camp etal. (1996) Plant Physiol. 112 (2): 525-535; Canevascini et al. (1996)Plant Physiol. 112 (2): 513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35 (5): 773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23 (6): 1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90 (20): 9586-9590.

A promoter can additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate. Using the promoter sequences disclosed here, it is possible toisolate and identify further regulatory elements in the 5′ regionupstream from the particular promoter region identified. Thus thepromoter region disclosed is generally further defined by comprisingupstream regulatory elements such as those responsible for high leveland temporal expression of the coding sequence, enhancers and the like.In the same manner, the promoter elements which enable low to high levelexpression can be identified, isolated, and used with other corepromoters to confirm embryo-preferred expression. By core promoter ismeant the sequence sometimes referred to as the TATA box (or similarsequence) which is common to promoters in most genes encoding proteins.Thus the upstream promoter of the promoter can optionally be used inconjunction with its own or core promoters from other sources.

Any plant promoter can be used as a 5′ regulatory element for modulatingexpression of a particular gene or genes operably associated thereto.When operably linked to a transcribable polynucleotide molecule, apromoter typically causes the transcribable polynucleotide molecule tobe transcribed in a manner that is similar to that of which the promoteris normally associated. Plant promoters can include promoters producedthrough the manipulation of known promoters to produce artificial,chimeric, or hybrid promoters. Such promoters can also combinecis-elements from one or more promoters, for example, by adding aheterologous regulatory element to an active promoter with its ownpartial or complete regulatory elements. Thus, the design, construction,and use of chimeric or hybrid promoters comprising at least onecis-element of SEQ ID NOs: 1, 2, 3, or 4 for modulating the expressionof operably linked polynucleotide sequences is encompassed by thepresent invention.

As used herein, the term “cis-element” refers to a cis-actingtranscriptional regulatory element that confers an aspect of the overallcontrol of gene expression. A cis-element may function to bindtranscription factors, trans-acting protein factors that regulatetranscription. Some cis-elements bind more than one transcriptionfactor, and transcription factors may interact with different affinitieswith more than one cis-element. The promoters of the present inventiondesirably contain cis-elements that can confer or modulate geneexpression. Cis-elements can be identified by a number of techniques,including deletion analysis, i.e., deleting one or more nucleotides fromthe 5′ end or internal to a promoter; DNA binding protein analysis usingDNase I footprinting, methylation interference, electrophoresismobility-shift assays, in vivo genomic footprinting by ligation-mediatedPCR, and other conventional assays; or by DNA sequence similarityanalysis with known cis-element motifs by conventional DNA sequencecomparison methods. The fine structure of a cis-element can be furtherstudied by mutagenesis (or substitution) of one or more nucleotides orby other conventional methods. Cis-elements can be obtained by chemicalsynthesis or by isolation from promoters that include such elements, andthey can be synthesized with additional flanking nucleotides thatcontain useful restriction enzyme sites to facilitate subsequencemanipulation.

As used herein, a nucleotide segment is referred to as operably linkedwhen it is placed into a functional relationship with another nucleotidesegment. For example, DNA for a signal sequence is operably linked toDNA encoding a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it stimulates thetranscription of the sequence. Generally, nucleic acid molecules thatare operably linked are contiguous, and in the case of a signal sequenceboth contiguous and in reading phase. However, enhancers need not becontiguous with the coding sequences whose transcription they control.Linking is accomplished by ligation at convenient restriction sites orat adapters or linkers inserted in lieu thereof. The expression cassettecan include one or more enhancers in addition to the promoter. Byenhancer is intended a cis-acting sequence that increases theutilization of a promoter. Such enhancers can be native to a gene orfrom a heterologous gene. Further, it is recognized that some promoterscan contain one or more native, enhancers or enhancer-like elements. Anexample of one such enhancer is the 35S enhancer, which can be a singleenhancer, or duplicated. See for example, McPherson et al, U.S. Pat. No.5,322,938.

The promoters of the invention may be combined with any number of othercomponents to be introduced into the plant, including combined with anucleotide sequence of interest to be expressed in the plant. The“nucleotide sequence of interest” refers to a nucleotide sequence thatencodes for a desired polypeptide or protein but also may refer tonucleotide sequences that do not constitute an entire gene, and which donot necessarily encode a polypeptide or protein. For example, when usedin a homologous recombination process, the promoter may be placed in aconstruct with a sequence that targets an area of the chromosome in theplant but may not encode a protein. Use of antisense versions of anucleic acid sequence is another example where use of a sequence may notresult in an encoded protein. If desired, the nucleotide sequence ofinterest can be optimized for plant translation by optimizing the codonsused for plants and the sequence around the translational start site forplants. Sequences resulting in potential mRNA instability can also beavoided.

In general, the methods available for construction of recombinant genes,optionally comprising various modifications for improved expression, candiffer in detail. However, conventionally employed methods include PCRamplification, or the designing and synthesis of overlapping,complementary synthetic oligonucleotides, which are annealed and ligatedtogether to yield a gene with convenient restriction sites for cloning,or subcloning from another already cloned source, or cloning from alibrary. The methods involved are standard methods for a molecularbiologist (Sambrook et al., 1989 supra). An expression vector is a DNAmolecule comprising a gene or antisense DNA that is expressed in a hostcell. Typically, gene expression is placed under the control of certainregulatory elements, including constitutive or inducible promoters,tissue-specific regulatory elements, and enhancers.

One skilled in the art readily appreciates that the promoter can be usedwith any of a variety of nucleotide sequences comprising the nucleotidesequence of interest to be expressed in plants. In referring to anoperably linked nucleotide sequence is intended a functional linkagebetween a promoter and another sequence where the promoter initiates andmediates transcription of the nucleotide sequence. For example, thenucleotide sequence of interest may encode a protein that is useful forindustrial or pharmaceutical purposes or the like, or to impact theplant itself, such as through expression of a protein that providesdisease resistance, insect resistance, herbicide resistance, or impactsagronomic traits as well as grain quality traits. DNA sequences nativeto plants as well as non-native DNA sequences can be transformed intoplants and used to modulate levels of native or non-native proteins. Oneor more of such sequences and/or expression cassettes may be transformedinto a plant cell (in referring to a plant cell, it is intended toinclude cells without plant membranes, such as protoplasts).

Such nucleotide sequences include, but are not limited to, thoseexamples provided below:

1. Genes or Coding Sequence that Confer Resistance to Pests or Disease

(A) Plant Disease Resistance Genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium falvum (Jones et al., 1994 Science266:789), tomato Pto gene, which encodes a protein kinase, forresistance to Pseudomonas syringae pv. tomato (Martin et al., 1993Science 262:1432), and Arabidopsis RSSP2 gene for resistance toPseudomonas syringae (Mindrinos et al., 1994 Cell 78:1089).

(B). A B. thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon, such as, a nucleotide sequence of a B.thuringiensis δ-endotoxin gene (Geiser et al., 1986 Gene 48:109), and avegetative insecticidal (VIP) gene (see, e.g., Estruch et al. (1996)Proc. Natl. Acad. Sci. 93:5389-94). Moreover, DNA molecules encodingδ-endotoxin genes can be purchased from American Type Culture Collection(Rockville, Md.), under ATCC accession numbers. 40098, 67136, 31995 and31998.

(C) A lectin, such as, nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al., 1994 Plant Molec. Biol.24:825).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include, a rice cysteine proteinaseinhibitor (Abe et al., 1987 J. Biol. Chem. 262:16793), a tobaccoproteinase inhibitor I (Huub et al., 1993 Plant Molec. Biol. 21:985),and a α-amylase inhibitor Sumitani et al., 1993 Biosci. Biotech.Biochem. 57:1243).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof, such as, baculovirus expression of clonedjuvenile hormone esterase, an inactivator of juvenile hormone (Hammocket al., 1990 Nature 344:458).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. J. Biol. Chem. 269:9Examples of such genes include, an insect diuretic hormone receptor(Regan, 1994), an allostatin identified in Diploptera punctata (Pratt,1989) Biochem. Biophys. Res. Comm. 163:1243, insect-specific, paralyticneurotoxins (U.S. Pat. No. 5,266,361).

(H) An insect-specific venom produced in nature by a snake, a wasp,etc., such as, a scorpion insectotoxic peptide (Pang, 1992 Gene116:165).

(I) An enzyme responsible for a hyperaccumulation of monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al., 1993 Insect Molec. Biol.23:691) and parsley ubi4-2 polyubiquitin gene (Kawalleck et al., 1993Plant Molec. Biol. 21:673).

(K) A molecule that stimulates signal transduction. Examples of suchmolecules include, nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al., 1994 Plant Molec. Biol. 24:757) and a nucleotidesequence of a maize calmodulin cDNA clone (Griess et al., 1994 PlantPhysiol. 104:1467).

(L) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914, the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(M) A membrane permease, a channel former or a channel blocker, such as,a cecropin-β lytic peptide analog (Jaynes et al., 1993 Plant Sci. 89:43)which renders transgenic tobacco plants resistant to Pseudomonassolanacearum.

(N) A viral-invasive protein or a complex toxin derived there from. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al. (1990) Ann. Rev. Phytopathol. 28:451.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Forexample, Taylor et al. (1994) Abstract #497, Seventh Int'l. Symposium onMolecular Plant-Microbe Interactions shows enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.

(P) A virus-specific antibody. See, for example, Tavladoraki et al.(1993) Nature 266:469, which shows that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(O) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (Lamb et al., 1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992 Plant J. 2:367).

(R) A developmental-arrestive protein produced in nature by a plant,such as, the barley ribosome-inactivating gene has an increasedresistance to fungal disease (Longemann et al., 1992). Bio/Technology10:3305

(S) RNA interference in which an RNA molecule is used to inhibitexpression of a target gene. An RNA molecule in one example is partiallyor fully double stranded which triggers a silencing response resultingin cleavage of dsRNA into small interfering RNAs, which are thenincorporated into a targeting complex that destroys homologous mRNAs.See, e.g., Fire et al., U.S. Pat. No. 6,506,559; Graham et al. U.S. Pat.No. 6,573,099.

2. Genes that Confer Resistance to a Herbicide

(A) Genes encoding resistance or tolerance to a herbicide that inhibitsthe growing point or meristem, such as an imidazalinone or asulfonylurea. Exemplary genes in this category code for mutantacetolactate synthase (ALS) (Lee et al., 1988 EMBO J. 7:1241) also knownas acetohydroxyacid synthase (AHAS) enzyme (Miki et al., 1990 Theor.Appl. Genet. 80:449).

(B) One or more additional genes encoding resistance or tolerance toglyphosate imparted by mutant EPSP synthase and aroA genes, or throughmetabolic inactivation by genes such as GAT (glyophosateacetyltrasnferase or GOX (glyphosate oxidase) and other phosphonocompounds such as glufosinate (PAT and bar genes), and pyridinoxy orphenoxy proprionic acids and cyclohexadiones (ACCase inhibitor encodinggenes). See, for example, U.S. Pat. No. 4,940,835, which discloses thenucleotide sequence of a form of EPSPS which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061. European patentapplication No. 0 333 033 and U.S. Pat. No. 4,975,374 disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricinacetyl-transferase gene is provided inEuropean application No. 0 242 246. De Greef et al. (1989)Bio/Technology 7:61 describes the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexadiones, such as sethoxydim andhaloxyfop, are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al. (1992) Theor. Appl. Genet. 83:435.

(C) Genes encoding resistance or tolerance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla et al. (1991) Plant Cell 3:169describes the use of plasmids encoding mutant psbA genes to transformChlamydomonas. Nucleotide sequences for nitrilase genes are disclosed inU.S. Pat. No. 4,810,648, and DNA molecules containing these genes areavailable under ATCC accession numbers 53435, 67441 and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al. (1992) Biochem. J. 285:173.

(D) Genes encoding resistance or tolerance to a herbicide that binds tohydroxyphenylpyruvate dioxygenases (HPPD), enzymes which catalyze thereaction in which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. This includes herbicides such as isoxazoles (EP418175,EP470856, EP487352, EP527036, EP560482, EP682659, U.S. Pat. No.5,424,276), in particular isoxaflutole, which is a selective herbicidefor maize, diketonitriles (EP496630, EP496631), in particular2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2-phenyl)propane-1,3-dione,triketones (EP625505, EP625508, U.S. Pat. No. 5,506,195), in particularsulcotrione, or else pyrazolinates. A gene that produces anoverabundance of HPPD in plants can provide tolerance or resistance tosuch herbicides, including, for example, genes described at U.S. Pat.Nos. 6,268,549 and 6,245,968 and US publication No. 20030066102.

(E) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as, 2,4-dichlorophenoxyacetic acid (2,4-D) and which may alsoconfer resistance or tolerance to aryloxyphenoxypropionate (AOPP)herbicides. Examples of such genes include the α-ketoglutarate-dependentdioxygenase enzyme (AAD-1) gene, described at US Publication20090093366.

(F) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as, 2,4-dichlorophenoxyacetic acid (2,4-D) and which may alsoconfer resistance or tolerance to pyridyloxy auxin herbicides, such asfluoroxypyr or triclopyr. Examples of such genes include theα-ketoglutarate-dependent dioxygenase enzyme (AAD-12) gene, described atWO 2007/053482 A2.

(G) Genes encoding resistance or tolerance to dicambia (see, e.g., U.S.Patent Publication 20030135879).

(H) Genes providing resistance or tolerance to herbicides that inhibitprotoporphyrinogen oxidase (PPO) (see U.S. Pat. No. 5,767,373)

(I) Genes providing resistance or tolerance to triazine herbicides (suchas atrazine) and urea derivatives (such as diuron) herbicides which bindto core proteins of photosystem II reaction centers (PS II) (SeeBrussian et al., (1989) EMBO J. 1989, 8 (4): 1237-1245).

3. Genes that Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming maizeor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al., 1992) Proc.Nat. Acad. Sci. USA 89:2624.

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant, such asthe Aspergillus niger phytase gene (Van Hartingsveldt et al., 1993 Gene127:87).

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid (Raboy etal., 1990 Maydica 35:383).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al., 1988 J.Bacteriol. 170:810), Bacillus subtilis levansucrase gene (Steinmetz etal., 1985 Mol. Gen. Genel. 200:220), Bacillus licheniformis α-amylase(Pen et al., 1992 Bio/Technology 10:292), tomato invertase genes(Elliott et al., 1993 Plant Molec. Biol. 21:515), barley amylase gene(Sogaard et al., 1993 J. Biol. Chem. 268:22480), and maize endospermstarch branching enzyme II (Fisher et al., 1993 Plant Physiol.102:1045).

The nucleotide sequence of interest can also be a nucleotide sequenceused to target an area of the plant genome through homologousrecombination. The promoter may be placed in a construct with suchsequence, which sequence will not necessarily encode a protein. Thesequence recombines in the genome and the promoter may be placed at thedesired site targeted by the sequences to regulate the desiredendogenous nucleotide sequence.

Further, the promoter can be used to drive mRNA that can be used for asilencing system, such as are discussed supra.

A terminator region may also be included in the vector. An embodiment ofthe invention is the terminator sequence of the present invention, SEQID NO: 5. Alternatively, another terminator may be used in conjunctionwith the promoter of the invention. In referring to a terminatorsequence is meant a nucleotide sequence that signals the end oftranscription. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase (MacDonaldet al., 1991 Nuc. Acids Res. 19 (20) 5575-5581) and nopaline synthasetermination regions (Depicker et al., (1982) Mol. and Appl. Genet.1:561-573 and Shaw et al. (1984) Nucleic Acids Research Vol. 12, No. 20pp 7831-7846 (nos)). Examples of various other terminators include thepin II terminator from the protease inhibitor II gene from potato (An,et al. (1989) Plant Cell 1, 115-122. See also, Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidRes. 15:9627-9639.

In one embodiment, the expression vector also contains a nucleotidesequence encoding a selectable or scorable marker that is operably orfunctionally linked to a promoter that controls transcriptioninitiation, which can be the promoter of the invention or anotherpromoter. For a general description of plant expression vectors andreporter genes, see Gruber et al. (1993) Vectors for planttransformation. In: Glick, B. R. and Thompson J. E. (Eds.) Methods inPlant Molecular Biology and Biotechnology, CRC Press, pp. 89-119. Forexample, the selective gene is a glufosinate-resistance encoding DNA orphosphinothricin acetyl transferase (PAT) or a maize optimized PAT gene,or bar gene can be used under the control of the CaMV 35S or otherpromoter. Such PAT genes confer resistance to the herbicide bialaphos(Gordon-Kamm et al., 1990 Plant Cell 2, 603-618; Wohllenben et al. 1988Gene 70, 25-37). Other examples, without intending to be limiting, arehygromycin phosphotransferase, EPSP synthase and dihydropteroateencoding genes. (See Miki et al. (1993) “Procedures for IntroducingForeign DNA into Plants” in Methods in Plant Molecular Biology andBiotechnology Glick et al. (eds) CRC Press, pp. 67-88).

In addition, markers that facilitate identification of a plant cellcontaining the polynucleotide encoding the marker may be employed.Scorable or screenable markers are useful, where presence of thesequence produces a measurable product and can produce the productwithout destruction of the plant cell. Examples include aβ-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (for example, U.S. Pat. Nos.5,268,463 and 5,599,670); chloramphenicol acetyl transferase (Jeffersonet al. The EMBO Journal vol. 6 No. 13 pp. 3901-3907); alkalinephosphatase. In a preferred embodiment, the marker used is beta-caroteneor provitamen A (Ye et al, Science 287:303-305-(2000)). The gene hasbeen used to enhance the nutrition of rice, but in this instance it isemployed instead as a screenable marker, and the presence of the genelinked to a gene of interest is detected by the golden color provided.Unlike the situation where the gene is used for its nutritionalcontribution to the plant, a smaller amount of the protein is needed.Other screenable markers include the anthocyanin/flavonoid genes ingeneral (See discussion at Taylor and Briggs, The Plant Cell (1990)2:115-127) including, for example, a R-locus gene, which encodes aproduct that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., in Chromosome Structure andFunction, Kluwer Academic Publishers, Appels and Gustafson eds., pp.263-282 (1988)); the genes which control biosynthesis of flavonoidpigments, such as the maize C1 gene (Kao et al., Plant Cell (1996) δ:1171-1179; Scheffler et al. Mol. Gen. Genet. (1994) 242:40-48) and maizeC2 (Wienand et al., Mol. Gen. Genet. (1986) 203:202-207); the B gene(Chandler et al., Plant Cell (1989) 1:1175-1183), the p1 gene (Grotewoldet al, Proc. Natl. Acad. Sci. USA (1991) 88:4587-4591; Grotewold et al.,Cell (1994) 76:543-553; Sidorenko et al., Plant Mol. Biol. (1999)39:11-19); the bronze locus genes (Ralston et al., Genetics (1988)119:185-197; Nash et al., Plant Cell (1990) 2 (11): 1039-1049), amongothers. Yet further examples of suitable markers include the cyanfluorescent protein (CYP) gene (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), the yellowfluorescent protein gene (PhiYFP™ from Evrogen; see Bolte et al. (2004)J. Cell Science 117: 943-54); a lux gene, which encodes a luciferase,the presence of which may be detected using, for example, X-ray film,scintillation counting, fluorescent spectrophotometry, low-light videocameras, photon counting cameras or multiwell luminometry (Teeri et al.(1989) EMBO J. 8:343); a green fluorescent protein (GFP) gene (Sheen etal., Plant J. (1995) 8 (5):777-84); and DsRed2 where plant cellstransformed with the marker gene are red in color, and thus visuallyselectable (Dietrich et al. (2002) Biotechniques 2 (2):286-293).Additional examples include a p-lactamase gene (Sutcliffe, Proc. Nat'l.Acad. Sci. U.S.A. (1978) 75:3737), which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l. Acad. Sci.U.S.A. (1983) 80:1101), which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech.(1990) 8:241); and a tyrosinase gene (Katz et al., J. Gen. Microbiol.(1983) 129:2703), which encodes an enzyme capable of oxidizing tyrosineto DOPA and dopaquinone, which in turn condenses to form the easilydetectable compound melanin. Clearly, many such markers are available toone skilled in the art.

The expression vector can optionally also contain a signal sequencelocated between the promoter and the gene of interest and/or after thegene of interest. A signal sequence is a nucleotide sequence, translatedto give an amino acid sequence, which is used by a cell to direct theprotein or polypeptide of interest to be placed in a particular placewithin or outside the eukaryotic cell. One example of a plant signalsequence is the barley α-amylase secretion signal (Rogers, 1985 J. Biol.Chem. 260, 3731-3738). Many signal sequences are known in the art. See,for example Becker, T. W., Templeman, T. S., Viret, J. F. and Bogorad,L. (1992) The cab-m7 gene: a light-inducible, mesophyll-specific gene ofmaize. Plant Mol. Biol. 20, 49-60; Fontes, et al. (1991)Characterization of an immunoglobulin binding protein homolog in themaize floury-2 endosperm mutant. Plant Cell 3, 483-496; Matsuoka, K. andNakamura, K. (1991) Propeptide of a precursor to a plant vacuolarprotein required for vacuolar targeting. Proc. Natl. Acad. Sci. USA 88,834-838; Gould et al. (1989) A conserved tripeptide sorts proteins toperoxisomes. J. Cell. Biol. 108, 1657-1664; Creissen et al. (1992)Molecular characterization of glutathione reductase cDNA from pea (Pisumsativum L.). Plant J. 2, 129-131; Kalderon et al. (1984) A short aminoacid sequence able to specify nuclear location. Cell 39, 499-509 andStiefel et al. (1990) Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation. Plant Cell 2, 785-793.

Leader sequences can be included to enhance translation. Variousavailable leader sequences may be substituted or added. Translationleaders are known in the art and include, for example: picornavirusleaders, for example, EMCV leader (encephalomyocarditis 5′ noncodingregion) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco EtchVirus) (Gallie et al. (1995) Gene 165 (2):233-8); human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature353:90-94); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV) (Gallie. (1987) Nucleic Acids Res. 15(8):3257-73); and maize chlorotic mottle virus leader (MCMV) (Lommel etal. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987)Plant Physiology 84:965-968. Other methods known to enhance translationcan also be utilized, for example, introns, and the like. Obviously,many variations on the promoters, selectable markers, signal sequences,leader sequences, termination sequences, introns, enhancers and othercomponents of the vector are available to one skilled in the art.

Where appropriate, the nucleotide sequence (s) may be optimized forincreased expression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) (1990) Plant Physiol. 92: 1-11for a discussion of host-preferred codon usage. Methods are available inthe art for synthesizing plant-preferred genes. See, for example, U.S.Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498. Additional sequence modifications are known to enhancegene expression in a plant. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. When possible, the sequence is modified to avoid predicted hairpinsecondary mRNA structures.

In preparing the nucleotide construct, the various nucleotide sequencefragments can be manipulated, so as to provide for the nucleotidesequences in the proper orientation and, as appropriate, in the properreading frame. Toward this end, adapters or linkers can be employed tojoin the nucleotide sequence fragments or other manipulations may beinvolved to provide for convenient restriction sites, removal ofsuperfluous nucleotide sequences, removal of restriction sites, or thelike. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. (See, forexample, Miki and McHugh (2004) Biotechnol. 107, 193-232; Klein et al.(1992) Biotechnology (N Y) 10, 286-291; and Weising et al. (1988) Annu.Rev. Genet. 22, 421-477). For example, the DNA construct may beintroduced into the genomic DNA of the plant cell using techniques suchas microprojectile-mediated delivery (Klein et al. 1992, supra),electroporation (Fromm et al., 1985 Proc. Natl. Acad. Sci. USA 82,5824-5828), polyethylene glycol (PEG) precipitation (Mathur and Koncz,1998 Methods Mol. Biol. 82, 267-276), direct gene transfer (WO 85/01856and EP-A-275 069), in vitro protoplast transformation (U.S. Pat. No.4,684,611), and microinjection of plant cell protoplasts or embryogeniccallus (Crossway, A. (1985) Mol. Gen. Genet. 202, 179-185).Agrobacterium transformation methods of Ishida et al. (1996) and alsodescribed in U.S. Pat. No. 5,591,616 are yet another option.Co-cultivation of plant tissue with A. tumefaciens is a variation, wherethe DNA constructs are placed into a binary vector system (Ishida etal., 1996 Nat. Biotechnol. 14, 745-750). The virulence functions of theA. tumefaciens host will direct the insertion of the construct into theplant cell DNA when the cell is infected by the bacteria. See, forexample, Fraley et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 4803-4807.Agrobacterium is primarily used in dicots, but monocots including maizecan be transformed by Agrobacterium. See, for example, U.S. Pat. No.5,550,318. In one of many variations on the method, Agrobacteriuminfection of corn can be used with heat shocking of immature embryos(Wilson et al. U.S. Pat. No. 6,420,630) or with antibiotic selection ofType II callus (Wilson et al., U.S. Pat. No. 6,919,494).

Rice transformation is described by Hiei et al. (1994) Plant J. 6,271-282 and Lee et al. (1991) Proc. Nat. Acad. Sci. USA 88, 6389-6393.Standard methods for transformation of canola are described by Moloneyet al. (1989) Plant Cell Reports 8, 238-242. Corn transformation isdescribed by Fromm et al. (1990) Biotechnology (N Y) 8, 833-839 andGordon-Kamm et al. (1990) supra. Wheat can be transformed by techniquessimilar to those used for transforming corn or rice. Sorghumtransformation is described by Casas et al. (Casas et al. (1993)Transgenic sorghum plants via microprojectile bombardment. Proc. Natl.Acad. Sci. USA 90, 11212-11216) and barley transformation is describedby Wan and Lemaux (Wan and Lemaux (1994) Generation of large numbers ofindependently transformed fertile barley plants. Plant Physiol. 104,37-48). Soybean transformation is described in a number of publications,including U.S. Pat. No. 5,015,580.

In one preferred method, use of aerosol beam technology for introductionof nucleotide sequences into cells is employed. Aerosol beam technologyemploys the jet expansion of an inert gas as it passes from a region ofhigher gas pressure to a region of lower gas pressure through a smallorifice. The expanding gas accelerates aerosol droplets containing themolecules to be introduced into a cell or tissue. Aerosol dropletsproduced are typically less than 0.1 micron in diameter at the point ofimpact with the target cells. DNA carried in aerosol droplets of thissmall size penetrates cells only because of the speeds attained by theaerosol droplets. Speeds achieved by the aerosol beam method of theinvention are supersonic and can reach 2000 meters/second. In apreferred embodiment, the process includes (I) culturing a source ofcells, (II) optionally, pretreating cells to yield tissue with increasedcapacity for uptake and integration by aerosol beam technology, (III)transforming said tissue with an exogenous nucleotide sequence by theaerosol beam method of the invention, (IV) optionally, identifying orselecting for transformed tissue, (V) optionally, regeneratingtransgenic plants from the transformed cells or tissue, and (VI)optionally, producing progeny of said transgenic plants. This process isdescribed in detail at Held et al., U.S. Pat. Nos. 6,809,232; 7,067,716;and 7,026,286 (all incorporated herein by reference in their entirety).

In accordance with the present invention, a transgenic plant can beproduced that contains an introduced non-pollen preferred promoter. Itcan be combined with any one of the components set forth above.

In a further embodiment, plant breeding can be used to introduce thenucleotide sequences into other plants once transformation has occurred.This can be accomplished by any means known in the art for breedingplants such as, for example, cross pollination of the transgenic plantsthat are described above with other plants, and selection for plantsfrom subsequent generations which contain the nucleic acid and/orexpress the amino acid sequence or trait. The plant breeding methodsused herein are well known to one skilled in the art. For a discussionof plant breeding techniques, see Poehlman, J. M. and Sleper, D. A.(1995) Breeding Field Crops, 4th Edition, Iowa State University Press.Many crop plants useful in this method are bred through techniques thattake advantage of the plant's method of pollination. A plant isself-pollinating if pollen from one flower is transferred to the same oranother flower of the same plant. A plant is cross-pollinating if thepollen comes from a flower on a different plant. For example, inBrassica, the plant is normally self-sterile and can only becross-pollinated unless, through discovery of a mutant or throughgenetic intervention, self-compatibility is obtained. Inself-pollinating species, such as rice, oats, wheat, barley, peas,beans, soybeans, tobacco and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower. Maize plants (Zea mays L.) can be bred byboth self-pollination and cross-pollination techniques. Maize has maleflowers, located on the tassel, and female flowers, located on the ear,on the same plant. It can self or cross-pollinate.

Pollination can be by any means, including but not limited to hand, windor insect pollination, or mechanical contact between the male fertileand male sterile plant. For production of hybrid seeds on a commercialscale in most plant species pollination by wind or by insects ispreferred. Stricter control of the pollination process can be achievedby using a variety of methods to make one plant pool male sterile, andthe other the male fertile pollen donor. This can be accomplished byhand detassling, cytoplasmic male sterility, or control of malesterility through a variety of methods well known to the skilledbreeder. Examples of more sophisticated male sterility systems includethose described by Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219and Albertsen et al., U.S. Pat. Nos. 5,859,341 and 6,013,859.

Backcrossing methods may be used to introduce the gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPoehlman et al. (1995) supra.

Further, the plant, seed or tissue can be further processed into a plantproduct, such as including grain products such as flour, meal, andgrits, or separated starches, oil, protein and oil and the like.

EXAMPLES

The following is presented as illustrative of an embodiment of theinvention and does not limit the scope of the invention as otherwise setforth.

Example 1 Soybean Promoters Lacking Expression in Pollen

A screening was undertaken to identify promoters that lack expression insoybean pollen using gene-chip analysis. To achieve this goal, total RNAfrom leaf, root, and pollen tissues was isolated using ‘Plant RNeasyKit’ of Qiagen. Duplicate preparations were made from each of thesetissues on 2 different days. Over 30 μg of highly pure RNA samples fromeach tissue type were obtained from each replicate preparation. Thesesamples were submitted to the Iowa State University Gene-Chip facilityfor analysis. Analysis of soybean gene expression for each tissue typewas done in duplicate. A total of about 38,000 gene data points werecollected per chip. From the results four gene candidates were chosenexpressed at low or no levels in pollen but were expressed in the leavesand roots. These candidates were called GSO, GNR, 185, and 17. Thecomplete sequences of these genes including their promoter andterminator sequences were obtained from the recently published soybeangenome database(www.phytozome.net/search.php?show=blast&blastdb=soybean, University ofCalifornia Regents, Center for Integrative Genetics, 2010). Thisinformation was used in designing the following specific primers toamplify the promoter regions:

GNR: (SEQ ID NO: 6) GATCTTCATTTATCCATTGGGGTACTTGTTTC (SEQ ID NO: 7)TGAGGAAATTAAATGAAAGGAAAAGAAAATTAGAG GSO: (SEQ ID NO: 8)GAAGGAGATCTAGTTCACTGGTTAAATAAGATGTG (SEQ ID NO: 9)GGTCTACTGAGGCGTGTGGCTGGAGTGAGG GNRter: (SEQ ID NO: 10)TTAACCAGTGCATGATGCTGAATTAAATG (SEQ ID NO: 11) TTATAATGTAGTTTCAACTTGAATCC17: (SEQ ID NO: 12) ATCCCATGGTCGCGGACGATGTAATAGAAC (SEQ ID NO: 13)ATCGGATCCACCCGCCCATACATCGTAACCAC 185: (SEQ ID NO: 14)ACTGCGGCCGCGAGTATGACCCTTGATGCCGC (SEQ ID NO: 15)ACTGGATCCACCTTAGTTAGGATTTTGTGTTTT

Using the soybean Jack [See, Nickell, C. D., G. R. Noel, D. J. Thomas,and R. Waller (1990) Registration of ‘Jack’ soybean. Crop Sci 1365. 30]genomic DNA as a template and the specific primers, promoters wereamplified by PCR. The amplicons were cloned in pGEM-T Easy vector andcorrect clones containing the GNR, GSO, 17, and 185 inserts wereidentified by restriction digestion analyses. The nucleotide sequencesof the cloned GNR (FIG. 1), GSO (FIG. 2), 17 (FIG. 3) and 185 (FIG. 4)promoters were determined. Comparison (GAP) of these sequences to theGNR, GSO, 17, and 185 sequences reported in the ‘soybean genomedatabase’ revealed that the cloned promoter sequences were >99.5%identical to the reported sequences.

The amplified GNR and GSO promoters cloned in pGEM-TEasy vectors wereexcised as EcoRI/BamHI fragments (1065 bp GNR fragment and 890 bp GSOfragment). These fragments were introduced into an EcoRI/BamHI digestedplasmid vector. Subsequently, the GNR terminator (FIG. 5) was introducedinto to these constructs (as AscI/SacI fragments) to generatepGNRproGNRter and pGSOproGNRter constructs.

A GUS gene was amplified as a BamHI/AscI fragment through PCR and theamplicon was cloned into pGEM-T vector. A correct pGEM-T/GUS clone wasidentified by restriction digestion analysis. The GUS insert from thisclone was excised as a BamHI/AscI fragment and was introduced intoBamHI/AscI-digested pGNRproGNRter and pGSOproGNRter backbones togenerate pGNRproGUSGNRter and pGSOproGUSGNRter constructs, respectively(FIGS. 6 & 7). The pGNRproGUSGNRter and pGSOproGUSGNRter constructs werelinearized with EcoRI and SalI. The NPTII cassette (35S NPTII Ocs) wasremoved as an EcoRI and SalI fragment and introduced into theEcoRI/SalI-digested pGNRproGUSGNRter and pGSOproGUSGNRter constructs togenerate pGNRGUSNPT and pGSOGUSNPT, respectively (FIGS. 8 & 9). Theseconstructs were used in the transformation of soybean.

Promoters 17 and 185 were amplified with PCR as is common in the art tofacilitate cloning them upstream of a GUS gene. Primers used to amplifypromoter 17 were (actcaattgacccgcccatacatcgtaaccactata) (SEQ ID NO: 16)and (atcggatcccgcggacgatgtaatagaactagctag) (SEQ ID NO: 17). Primers usedto amplify promoter 185 were (actcaattggagtatgacccttgatgccgccaag) (SEQID NO: 18) and (actggatccaccttagttaggattttgtgttttgagtg) (SEQ ID NO: 19).The amplified bands were cloned into pGEM-T Easy vector according to themanufacturer Promega. The 17 and 185 promoters in pGEM-T were digestedwith MfeI and BamHI and cloned into the EcoRI and BamHI sites ofpGSOproGUSGNRterNPT, thus, replacing the GSO promoter with the 17 and185 promoters, respectively. The resulting constructs were called17GUSNPT (FIG. 10) and 185GUSNPT (FIG. 11). These constructs were usedin the transformation of soybean. Soybean was transformed according tothe procedure described in U.S. Pat. No. 6,809,232, incorporated hereinby reference in its entirety.

Transgenic plant tissue was evaluated for GUS expression using ahistochemical staining procedure. Leaf tissue and pollen were incubatedin the presence of the substrate X-gluc (Gold Biotechnology, Inc.) at aconcentration of 0.5 mg/ml in 0.1 M sodium phosphate buffer pH 7.0 and0.1% Triton-x-100 at 37.degree. C for 1-8 hours. Plants were obtainedwhich expressed GUS in the leaves but no detectable expression in thepollen for the 17, 185, GNR and GSO promoters.

1. An isolated polynucleotide comprising a polynucleotide selected fromthe group consisting of: (a) a nucleotide sequence comprising SEQ ID NO:1; (b) a nucleotide sequence comprising SEQ ID NO: 2; (c) a nucleotidesequence comprising SEQ ID NO: 3; (d) a nucleotide sequence comprisingSEQ ID NO: 4; (e) a nucleotide sequence having at least 95% identity toany of sequences in parts (a)-(d), wherein said sequence has promoterfunction; (f) a nucleotide sequence having at least 96% identity to anyof sequences in parts (a)-(d,) wherein said sequence has promoterfunction; (g) a nucleotide sequence having at least 97% identity to anyof sequences in parts (a)-(d), wherein said sequence has promoterfunction; (h) a nucleotide sequence having at least 98% identity to anyof sequences in parts (a)-(d), wherein said sequence has promoterfunction; (i) a nucleotide sequence having at least 99% identity to anyof sequences in parts (a)-(d), wherein said sequence has promoterfunction; (j) a polynucleotide which is complementary to any ofsequences in parts (a)-(d); and (k) a fragment of any of sequences inparts (a)-(d), wherein said fragment has promoter function saidpolynucleotide operably linked to a heterologous transcribable nucleicacid molecule.
 2. The polynucleotide of claim 1, wherein saidpolynucleotide is operably linked to the polynucleotide of SEQ ID NO: 5.3. A plant cell comprising the polynucleotide of claim
 1. 4. A plantcomprising the polynucleotide of claim
 1. 5. Seed of a plant comprisingthe polynucleotide of claim
 1. 6. Grain product produced from the seedof claim
 5. 7. An isolated polynucleotide comprising a polynucleotideselected from the group consisting of: (a) a nucleotide sequencecomprising SEQ ID NO: 1; (b) a nucleotide sequence comprising SEQ ID NO:2; (c) a nucleotide sequence comprising SEQ ID NO: 3; and (d) anucleotide sequence comprising SEQ ID NO:
 4. 8. A method of expressing aheterologous nucleic acid molecule in a plant, the method comprisingintroducing into said plant a heterologous nucleic acid moleculeoperably linked to a promoter comprising a polynucleotide selected fromthe group consisting of: (a) a nucleotide sequence comprising SEQ ID NO:1; (b) a nucleotide sequence comprising SEQ ID NO: 2; (c) a nucleotidesequence comprising SEQ ID NO: 3; (d) a nucleotide sequence comprisingSEQ ID NO: 4; (e) a nucleotide sequence having at least 95% identity toany of sequences in parts (a)-(d), wherein said sequence has promoterfunction; (f) a nucleotide sequence having at least 96% identity to anyof sequences in parts (a)-(d,) wherein said sequence has promoterfunction; (g) a nucleotide sequence having at least 97% identity to anyof sequences in parts (a)-(d), wherein said sequence has promoterfunction; (h) a nucleotide sequence having at least 98% identity to anyof sequences in parts (a)-(d), wherein said sequence has promoterfunction; (i) a nucleotide sequence having at least 99% identity to anyof sequences in parts (a)-(d), wherein said sequence has promoterfunction; (j) a polynucleotide which is complementary to any ofsequences in parts (a)-(d); and (k) a fragment of any of sequences inparts (a)-(d), wherein said fragment has promoter function.
 9. Themethod of claim 8, wherein said heterologous nucleic acid molecule isexpressed at lower levels in pollen cells of said plant than in othercells of said plant.
 10. The method of claim 8, wherein saidheterologous nucleic acid molecule encodes a polypeptide that conferstolerance to a herbicide.
 11. The method of claim 8, wherein saidheterologous nucleic acid molecule encodes a Bacillus thuringiensispolypeptide that is expressed at lower levels in pollen cells of saidplant than in other cells of said plant.
 12. The method of claim 8,further comprising producing a grain product from said plant.
 13. Aregulatory region comprising SEQ ID NO:
 5. 14. A method of regulatingexpression of a heterologous nucleic acid molecule comprising operablylinking said nucleic acid molecule to SEQ ID NO: 5.